Traumatic spinal cord injury occurs in approximately 10-12,000 people per year in the United States. Most of the victims will survive the first year, and many will lead healthy and productive lives, although they will likely require a significant amount of care to maintain their well being. Given this, a person having a spinal cord injury is eager for treatment paradigms to promote repair and regain mobility. However, spinal cord injury presents an extremely complex set of problems to overcome to promote healing and reinnervation.
It was long thought that the central nervous system (CNS) lacked the ability to regenerate after injury. However, the use of peripheral nerve grafts in the brain and spinal cord that promoted axonal growth disproved this theory (Aguayo et al., “Axonal Elongation in Peripheral and Central Nervous System Transplants”, Adv. Cell Neurobiol. 3:215-234, 1979; Benfey and Aguayo, “Extensive Elongation of Axons from Rat Brain into Peripheral Nerve Grafts”, Nature 296:150-152, 1982). Peripheral neurons show a rapid response to injury, upregulating proteins associated with axon elongation, and are capable of regeneration. This is likely due to the presence of factors in the environment that stimulate process elongation and supply a permissive substrate to guide axon regrowth. In contrast, damage to the CNS initiates glial cell infiltration and pronounced scar formation, which ultimately inhibits repair and regeneration. However, Aguayo's results demonstrated that given the right substrate, CNS neurons retain the capacity to regenerate. Thus, failure of the adult neurons in the CNS to regenerate can be attributed to the local microenvironment.
Since this initial discovery, much work has been dedicated to identify conditions that will stimulate and guide axon regrowth in the CNS. At present, it is generally recognized that a multifaceted approach will be necessary to optimize repair and re-establish connections. First, for any reinnervation to occur, neurons have to survive the initial acute trauma. Successful treatment of chronic SCI would require this population of cells to survive for long periods. Second, the afflicted population of neurons has to be responsive to signals in the environment that stimulate axon regrowth. This may be problematic in adult neurons, where intracellular signaling mechanisms change with age, making the cells more responsive to inhibitory molecules (Cai et al., “Neuronal Cyclic AMP Controls the Developmental Loss in Ability of Axons to Regenerate”, J. Neurosci. 21(13); 4731-4739, 2001). Third, there are a number of inhibitory molecules present in the lesion site that can actively block process elongation. Finally, the timing of any therapeutic treatment may also be important to optimize regrowth. It has been recently demonstrated that the acute immune response observed after injury is beneficial to the repair process. The infiltrating immune cells clear debris and secrete neuroprotective factors. Delayed administration of therapeutic treatments until after the peak immunological activity enhances neuronal survival and axonal regrowth (Coumans et al., “Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins”, J. Neurosci 21:9334-9344, 2001). Thus, any successful treatment paradigm that is developed to treat SCI (either acute or chronic cases) will need to accommodate all of these factors.
Since the local microenvironment in the adult CNS is not conducive to spontaneous repair, one approach to stimulate repair is to change the environment to a more permissive one. This generally involves grafting a permissive substrate at the lesion site. The transplantation of Schwann cells (Xu et al., “A combination of BDNF and NT-3 promotes supraspinal axonal regeneration into Schwann cell grafts in the adult rat spinal cord”, Exp. Neurol. 134:261-272, 1995), stem cells (Mc Donald et al., “Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord”, Nature Med. 5:1410-1412, 1999), or olfactory ensheathing cells (Li et al., “Regeneration of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells”, J Neurosci 18:10514-10524, 1998) have all been demonstrated to promote axonal elongation over the lesion site. The cells provide a positive substrate over which axons can grow, and likely secrete factors to promote survival and stimulate neurite outgrowth. Schwann cells, for example, are a rich source of extracellular matrix proteins and growth factors, and are the main reason that peripheral neurons can regenerate. However, in most studies, repair is limited; only a small percentage of axons have grown across the graft. There may be some behavioral improvement, but anatomically, it is difficult to show complete reinnervation of the targets. These findings suggest that other pharmacological approaches may be necessary to enhance recovery.
Another strategy involves promoting neuronal survival and axonal sprouting using neurotrophic and growth factors at the lesion site (Bregman et al., “Neurotrophic factors increase axonal growth after spinal cord injury and transplantation in the adult rat”, Exp. Neurol. 148: 475-494, 1997; Kobayashi et al., “BDNF and NT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stimulate GAP-43 and Ta1-tubuln mRNA expression and promote axonal regeneration”, J Neurosci. 17: 9583-9595, 1997; Liu et al., “Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rubrospinal axons and recovery of forelimb recovery”, J. Neurosci 19:4370-4387, 1999). However, these appear to work best when combined with graft implantation at the injury site and in fact, may be required to get elongation across the graft (Bregman et al., “Neurotrophic factors increase axonal growth after spinal cord injury and transplantation in the adult rat”, Exp. Neurol. 148: 475-494, 1997). It is hypothesized that neurotrophins aid the damaged neurons in the host, and stimulate cells in the graft as well. They, in turn, secrete more molecules that can promote process outgrowth. Neuronal populations are responsive to a number of neurotrophins, depending on their expression of the Trk neurotrophin receptors (reviewed by Miller and Kaplan, “On Trk for retrograde signaling”, Neuron 32(5):767-70, 2001). Infusion of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) can increase sprouting and elongation through grey matter, but not much growth is observed through white matter. Again, recovery of function is limited in these studies, and the anatomical analysis has not demonstrated significant regrowth.
One consideration in treating adult CNS injury is that the neurons themselves may not be very responsive to the environmental cues that are present. Guidance molecules such as semaphorins, netrins and BDNF can be either attractive or repulsive depending on the intracellular cyclic AMP levels (Song et al., “cAMP-induced switching in turning direction of nerve growth cones”, Nature 388: 275-279, 1997, Song et al., “Conversion of neuronal growth cones responses from repulsion to attraction by cyclic nucleotides”, Science 281:1515-1518, 1998). This is also true of myelin proteins such as myelin-associated glycoprotein (MAG), which has long been thought to inhibit neurite outgrowth. In fact, it is repulsive or attractive depending on the cyclic AMP concentration; if the cyclic AMP levels are high, MAG is a permissive substrate (Song et al., “Conversion of neuronal growth cones responses from repulsion to attraction by cyclic nucleotides”, Science 281:1515-1518, 1998). The important finding is that adult neurons are less likely to elevate their cyclic AMP levels as compared with young neurons; thus, adult neurons are inhibited by MAG (Cai et al., “Neuronal cyclic AMP controls the developmental loss in ability of axons to regenerate”, J Neurosci 21(13):4731-4739, 2001). Accordingly, changes in intracellular pathway may make it more difficult to stimulate axon outgrowth from adult neurons.
Another major reason for the failure of CNS neurons to regenerate is the abundance of molecules that inhibit neurite outgrowth. At present, there are two classes of inhibitory molecules that are largely responsible for this: myelin proteins, derived from damaged oligodendrocytes, and proteoglycans that comprise the glial scar.
Myelin contributes a number of proteins that have been shown to inhibit process outgrowth. The first to be identified was a protein called NogoA (reviewed by David and Lacroix, “Molecular approaches to spinal cord repair”, Ann. Rev Neurosci. 26:411-40, 2003), which is found on the surface of oligodendrocytes and some axons. Others that can contribute to inhibition are myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp), and the proteoglycan versican. These are all found in normal myelin, but when myelin is damaged, they are released at the injury site, and consequently restrict axon growth. Interestingly, they all signal through the receptor for Nogo, a protein called NgR. Thus, a reasonable strategy for enhancing repair is blocking the NgR receptor with an antibody, thus neutralizing the inhibitory molecules. Recent findings have shown that a blocking antibody to NogoA can enhance axonal sprouting and regrowth in the spinal cord after injury (Schnell and Schwab, “Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors”, Nature 343:269-72, 1990 and Schnell and Schwab, “Sprouting and regeneration of lesioned corticospinal tract fibers in the adult rat spinal cord”, Eur. J. Neurosci 5:1156-1172, 1993; Raineteau et al., “Spouting and regeneration after pyramidotomy and blockade of the myelin associated neurite growth inhibitors NI 35/250 in adult rats”, Eur J. Neurosci 11:1486-1490, 1999).
The other source of major inhibitory activity lies with the formation of a glial scar after CNS injury (reviewed by Morgenstern et al., “Chondroitin sulfate proteoglycans in the CNS injury response” Prog. Brain Res. 137:313-332, 2002). The scarring process is rapid and complex, and involves a number of cell types. Astrocytes, meningeal cells, oligodendrocyte precursors and microglia can invade the injury site, and upregulate the synthesis and secretion of chondroitin sulfate proteoglycans (CSPG). CSPG accumulation occurs very rapidly at the lesion site, generally within one-week post injury. There are several types of CSPG expressed after injury, and include NG2, neurocan, versican, phosphocan and brevican. All of these molecules consist of a protein core with large glycosaminoglycan (GAG) sugar side chains attached. It is believed that the GAG side chains are responsible for the major inhibitory effects on axon elongation, by blocking access to growth promoting molecules. If the GAG chains are removed by chondroitinase ABC cleavage (cABC), axons can grow on a CSPG substrate. This has been demonstrated both in vitro and in vivo, with significant axon regrowth (Zuo et al., “Degradation of chondroitin sulfate proteoglycan enhances the neurite-promoting potential of spinal cord tissue”, Exp. Neurol. 154:654-62, 1998; Moon et al, “regeneration of CNS axons back to their target following treatment of adult rat brain with chondroitinase ABC”, Nat. Neurosci. 4:465-6, 2001; Bradbury et al., “Chondroitinase ABC promotes functional recovery after spinal cord injury”, Nature 416:636-40, 2002). Thus, cABC administration is a promising treatment option for neutralizing inhibitors to axonal regeneration.
One limitation in the in vivo studies described above is the mode of application of the substances being tested as a treatment for SCI. Neurotrophins and cABC, for example, are often infused using repeated injections or mini-osmotic pumps. This mode of delivery may not be capable of delivering the optimal amounts of growth factor to the target site; for example, a large amount of neurotrophins pumped into a relatively wide area of the spinal cord may result in excessive sprouting, which, in turn, could lead to abnormal nerve connections. The duration and timing of treatments could also be problematic, especially if treatments are long term and need to change over time. A delivery system that is minimally invasive and that specifically targets the lesion site would be extremely useful in tailoring treatment therapies.
Loading molecules in biodegradable microspheres is one way to provide a sustained release in a localized target area. This has been applied with success to hormones (Johnson et el., “A month long effect from a single injection of microencapsulated human growth hormone”, Nature Medicine 2(7) 795, 1996), vaccines (Alonso et al., “Biodegradable microspheres as controlled release tetanus toxoid delivery systems”, Vaccine 12: 299-306, 1994) and chemotherapeutic agents (Rhines et al., “Local immunotherapy with interleukin-2 delivered from biodegradable polymer microspheres combined with interstitial chemotherapy: a novel treatment for experimental malignant glioma”, Neurosurgery 52(4):872-880, 2003). The composition can be varied to control the degradation time in vivo, and most conventional microspheres, such as polylactic/glycolic acid microspheres, have been show to be safe and effective in humans (Chaubal, “Polylactides/glycolides-excipients for injectible drug delivery and beyond”, Drug Del Tech 2:34-36, 2002). The development of techniques to generate nanospheres (average diameter of less than 1 micron) makes them an even more attractive drug delivery system, as they are more easily injected at the desired treatment site.
In view of the above, there is a need for providing a means to treat acute and chronic spinal cord injury at the site of the spinal cord injury itself. Specifically, a formulation is required that can be administered at the site of the SCI which is capable of substantially reducing (i.e., effectively inhibiting) scar formation in acute and chronic cases.